The present invention relates to nucleic acid sequences encoding members of the fibroblast growth factor (FGF) family, and to polypeptides encoded by the nucleic acid sequences.
The substantia nigra is an area of the brain that has generated intensive research. Interest in the substantia nigra was originally based on the finding that degeneration of dopaminergic neurons in the area causes Parkinson""s disease. In addition, the substantia nigra has been strongly implicated in thought and affective disorders (1). Therefore, neurotrophic factors for dopaminergic neurons in the substantia nigra are of substantial clinical interest.
Glial cell fine-derived neurotrophic factor (GDNF) is the first neurotrophic factor documented to enhance the survival of midbrain dopaminergic neurons (Lin, L.-F. H. et al., Science 260:1130-1132 (1993)). Persephin, Artemin, BDGF and NT-3 also enhance the survival of midbrain dopaminergic neurons and have clinical potential in the treatment of Parkinson""s disease (Milbrandt, J. et al., Neuron 20:245-253 (1998); Baloh,; R. H. et al., Neuron 21:1291-1302 (1998); Hyman, C. et al., Nature 350:230-232 (1991); Hyman, C. et al., J. Neurosci. 14:335-347 (1994)). However, GDNF was reported to be widely expressed in neurons of the brain (Pochon, N. A. et al., Eur. J. Neurosci. 9:463-471 (1997)). Persephin was also widely expressed in several major tissues including heart, kidney, liver and brain (Milbrandt, J. et al., Neuron 20:245-253 (1998)). Artemin in brain was expressed in the basal ganglia and thalamus, suggesting that it influences the subcortical motor system (Baloh, R. H. et al., Neuron 21:1291-1302 (1998)). BDNF and NT-3 were predominantly expressed in the hippocampus (Emfors, P. et al., Neuron 5:511-526 (1990)). Therefore, these neurotrophic factors appear not to be specific for dopaminergic neurons in the substantia nigra.
The prototypic fibroblast growth factors (FGFs), FGF-1 (aFGF) and FGF-2 (bFGF), were originally isolated from brain and pituitary as mitogens for fibroblasts. However, FGF-1 and FGF-2 are widely expressed in developing and adult tissues, and are polypeptides with multiple biological activities including angiogenesis, mitogenesis, cellular differentiation and repair of tissue injury (Baird, A. et al., Cancer Cells 3:239-243 (1991); Burgess, W. H. et al., Annu. Rev. Biochem. 58:575-606 (1989)). According to the published literature, the FGF family now consists of at least nineteen members, FGF-1 to FGF-19 (Dickson, C. et al., Ann. NY Acad. Sci. 638:18-26 (1991); Yoshida, T. et al., Ann. NY Acad. Sci. 638:27-37 (1991); Goldfarb, M. et al., Ann. NY Acad. Sci. 638:38-52 (1991); Coulier, F. et al., Ann. NY Acad. Sci. 638:53-61 (1991); Aaronson, S. A. et al., Ann. NY Acad. Sci. 638:62-77 (1991); Tanaka, A. et al., Proc. Natl. Acad. Sci. USA 89:8928-8932 (1992); Miyamoto, M. et al., Mol. Cell. Biol. 13:4251-4259 (1993); Yamasaki, M. et al., J. Biol. Chem. 271:15918-15921 (1996); Smallwood, P. M. et al., Proc. Natl. Acad Sci. USA 93:9850-9857 (1996); McWhirter, J. R. et al., Development 124:3221-3232 (1997); Miyake, A. et al., Biochem. Biophys. Res. Commun. 243:148-152 (1998); Hoshikawa, M. et al., Biochem. Biophys. Res. Commun. 244:187-191 (1998); Ohbayashi, N. et al., J. Biol. Chem. 273:18161-18164 (1998); Nishimura, T. et al., Biochim. Biophys. Acta 1444:148-151 (1999)). FGF-3 was identified to be a common target for activation by the mouse mammary tumor virus (Dickson, C. et al., Ann. NY Acad. Sci. 638:18-26 (1991)). FGF-4 to FGF-6 were identified as oncogene products (Yoshida, T. et al., Ann. NY Acad. Sci. 638:27-37 (1991); Goldfarb, M. et al., Ann. NY Acad. Sci. 638:38-52 (1991); Coulier, F. et al., Ann. NY Acad. Sci. 638:53-61 (1991)). FGF-10 was identified from rat lung by homology-based polymerase chain reaction (PCR) (Yamasaki, M. et al., J. Biol. Chem. 271:15918-15921 (1996)). FGF-11 to FGF-14 (FGF homologous factors (FHFs) 1 to 4) were identified from human retina by a combination of random cDNA sequencing, data base searches and homology-based PCR (Smallwood, P. M. et al., Proc. Natl. Acad. Sci. USA 93:9850-9857 (1996)). FGF-15 was identified as a downstream target of a chimeric homeodomain oncoprotein (McWhirter, J. R. et al., Development 124:3221-3232 (1997)). FGF-16, FGF-17, and FGF-18 were identified from rat heart and embryos by homology-based PCR, respectively (Miyake, A. et al., Biochem. Biophys. Res. Commun. 243:148-152 (1998); Hoshikawa, M. et al., Biochem. Biophys. Res. Commun. 244:187-191 (1998); Ohbayashi, N. et al., J. Biol. Chem. 273:18161-18164 (1998)). Recently, FGF-19 was identified from human fetal brain by data base search (Nishimura, T. et al., Biochim. Biophys. Acta 1444:148-151(1999)). They have a conserved xcx9c120-amino acid residue core with xcx9c30 to 60% amino acid identity. These FGFs also appear to play important roles in both developing and adult tissues. Thus, there is a need in the art for additional FGF molecules having functions and activities that differ from the known FGFs and for FGF molecules specifically expressed in regions of the brain implicated in human disease.
The present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising at least eight contiguous nucleotides of SEQ ID NO:1 or 3;
(b) a polynucleotide that encodes a variant of the polypeptide encoded by (a); and
(c) a polynucleotide encoding a protein expressed by a polynucleotide having the sequence of SEQ ID NO:1 or 3.
The invention further provides for the use of the isolated polynucleotides or fragments thereof as diagnostic probes or as primers.
The present invention also provides a composition comprising a polypeptide, wherein said polypeptide is selected from the group consisting of:
(a) a polypeptide comprising at least 6 contiguous amino acids encoded by SEQ ID NO:1 or 3;
(b) a polypeptide encoded by a polynucleotide comprising SEQ ID NO:1 or 3; and
(c) a variant of the polypeptide of (a) or (b).
Polypeptides of the invention are shown in SEQ ID NO:2 and 4. Other polypeptides comprise fragments of SEQ ID NO:2 and 4.
In certain preferred embodiments of the invention, the polynucleotide is operably linked to an expression control sequence. The invention further provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with the polynucleotide sequence. The invention also provides full-length cDNA and full-length polynucleotides corresponding to SEQ ID NO:1 or 3.
Protein and polypeptide compositions of the invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody that specifically reacts with such protein or polypeptide are also provided by the present invention.
The invention also provides for the production of large amounts of otherwise minor cell populations of cells to be used for generation of cDNA libraries for the isolation of rare molecules expressed in the precursors cell or progeny; cells produced by treatment may directly express growth factors or other molecules, and conditioned media is screened in assays for novel activities.
The invention further provides for the isolation, self-renewal and survival of mammalian neural stem cells and the differentiation of their progeny.
The invention also provides for compositions and methods of preventing or slowing degeneration of or increasing the numbers of dopaminergic neurons, such as in the substantial nigra, in disease states including Parkinson""s disease.
The invention further provides for compositions and methods of preventing or slowing degeneration of, or for enhancing the growth of, cells in the inner ear.